singeltons+contigs for 454 data

[litali]

Hi,
I am interested to create one file which will include the singeltons and contigs together, Is there any way to create such a file using 454 softwares or do I have to have a script which extracts the names of the singeltons from the readstatus file and then extracts the sequences from the fasta file and adds them to the contigs file?
Thanks alot!!!

[Torst]

I don't know of any script. I am replying to remind you that 454 sometimes uses PARTS of a read (the "left" end) and then puts the "right" end in 454ReadStatus.txt with the name "XXXXXX_right Singleton", so you'll need to think about what you want to do with those. eg.

% grep Singleton 454ReadStatus.txt
GHFU8EI02CHPMJ_left Singleton
GHFU8EI02B9FPY Singleton
GHFU8EI02CJNN4_right Singleton
GHFU8EI02CA0E9 Singleton

To get the .FASTA sequences from the .SFF file, you'll need to use "sffinfo":

% sffinfo -seq file.sff > file.fasta

Also, if you did paired end sequencing, the 454Scaffolds.fna file does NOT CONTAIN those contigs in 454Contigs.fna which failed to scaffold.

[westerman]

I thought that _left and _right should arise from paired end reads and not from split reads.

Basically what I do is to:

1) Grab the reads of choice from the 454ReadStatus.txt file and, optionally, the 454TrimStatus file

2) Use sfffile to create a temporary sff file with just those reads

3) Use sffinfo to extract the sequences.

The rough steps are:

fgrep '\tSingleton' 454ReadStatus.txt > /tmp/Singleton.tmp

sfffile -o /tmp/Singleton.sff /tmp/Singleton.tmp mysff.sff

sffinfo -s /tmp/Singleton.sff > Singleton.tfa

[flxlex]

Also, if you did paired end sequencing, the 454Scaffolds.fna file does NOT CONTAIN those contigs in 454Contigs.fna which failed to scaffold.

That is not entirely true: 454Scaffolds.txt contains the scaffolds (at least two contigs with gap(s)) AND all unscaffolded contigs of at least 2kb. IMO they shouldn't have done that, but rather outputted a separate unscaffolded-contig file...

sfffile -o /tmp/Singleton.sff /tmp/Singleton.tmp mysff.sff

I guess you mean

sfffile -o /tmp/Singleton.sff -i /tmp/Singleton.tmp mysff.sff

(note the '-i')

[Torst]

flxlex,

That is not entirely true: 454Scaffolds.txt contains the scaffolds (at least two contigs with gap(s)) AND all unscaffolded contigs of at least 2kb. IMO they shouldn't have done that, but rather outputted a separate unscaffolded-contig file...

Hmm, it appears you are correct. Thank you for replying! I had not noticed the "1 contig scaffolds" because, like you said, it is inconsistent and they get renamed to "scaffoldNNNNNN" ... but yes, when I examine 454Scaffolds.txt I can see many scaffolds which are made up of 1 contig only.. I find it hard to accept they would use a different threshold for "contigs becoming scaffolds" and "large contigs", and NOT output the separate unscaffolded contigs file too.

Also, you suggest the cut-off is 2kbp, but in my example 10 of the 22 contigs are between 1356bp and 1870bp, which suggests maybe the cutoff is 1kbp?

Either way - thank you muchly for catching my error!

[westerman]

I guess you mean

sfffile -o /tmp/Singleton.sff -i /tmp/Singleton.tmp mysff.sff

(note the '-i')

Yes, that is what I get for pulling the code out of a script that I use instead of typing it in directly. Thanks for the correction.